Fluorescent nitric oxide probes and associated methods

ABSTRACT

Nitric oxide probes including a compound represented by Formula, I, II, III, IV, V, VI or a combination thereof are provided. Methods of using these nitric oxide probes to detect nitric oxide are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/645,217, filed Oct. 4, 2012, which is a continuation ofPCT/US11/32226, filed Apr. 13, 2011 and claims priority to U.S.Provisional Application No. 61/326,750, filed Apr. 22, 2010, all ofwhich are incorporated herein by reference.

BACKGROUND

The biological roles of nitric oxide (NO) have led chemists andmolecular biologists to seek cellular imaging agents responsive to thisspecies. The creation of such agents derives from the pivotal role of NOin vasodilation as an endothelial-derived relaxing factor (EDRF), andits function as a platelet aggregation inhibitor, neurotransmitter,antimicrobial agent, and due to its antitumor activity incardiovascular, nervous, and immune systems. Although a variety ofquantification techniques have been developed, fluorescence techniquesare the most desirable because of their sensitivity, and highspatiotemporal resolution when combined with microscopy. Consequently, anumber of fluorescent NO probes are available, but each is hampered bycertain selectivity and/or synthetic limitations.

Currently, the most common approach for NO detection involves the use ofortho-diamino aromatics under aerobic conditions, which reacts with NO⁺equivalent, presumably N₂O₃ to furnish fluorescent triazole derivatives.Turn-on fluorescence signals are achieved due to suspension ofphotoinduced electron transfer (PET). Examples using fluoresceins (suchas DAF-2 DA), anthraquinones, rhodamines (such as DAR-4M AM), BODIPYs,and cyanines are documented. Such probes are among the current state ofthe art, yet severe limitations exist. First of all, in the presence ofH₂O₂/peroxidase, OONO⁻, OH., NO₂., and CO₃.⁻, the intrinsically electronrich diaminobenzene moiety is easily oxidized to an arylaminyl radical,which combines with NO and leads to triazoles. Second, dehydroascorbicacid (DHA) condenses with ortho-diamino aromatics and turns on thefluorescence of such probes. It was reported that 1 mM DHA yielded afluorescence signal with the commercial NO probes DAF-2 DA, DAR-4M AM,comparable to 300 nM and 100 μM of NO respectively. Third,benzotriazoles are pH sensitive (pK _(a)'s≈6.69) near neutral pH. The pHsensitivity can be solved by methylation of one of the amines, howeverthe reactivity of the probe toward DHA was undesirably enhanced.

The aforementioned limitations complicate NO detection usingortho-diamines Hence, a series of metal ligand complexes for NOdetection are also currently under development. For example,Cu^(II)(FL₅) , displays a fluorescence enhancement upon exposure to NOand can be used as a cellular imaging agent. However, given adissociation constant (K_(d)) of 1.5 μM and the presence other metalions in physiological conditions, it is a concern that the complex willrelease cytotoxic Cu²⁺. Complexes with lower K_(d)'s were reported,though with decreased reactivity toward NO.

Most recently, single-walled carbon nanotubes (SWCN) wrapped with3,4-diaminophenyl-functionalized dextrans were used for in vitro or invivo studies. The NIR fluorescence of the SWCN is bleached by severalreactive oxygen/nitrogen species, but at least NO does so more thanothers.

SUMMARY

The present disclosure generally relates to nitric oxide probes. Moreparticularly, the present disclosure relates to fluorescent nitric oxideprobes and associated methods.

The present disclosure provides a nitric oxide probe that may be used todetect and/or image NO and associated methods. In one embodiment, anitric oxide probe of the present disclosure comprises a compound thatis represented by the following Formula I:

wherein R₁ is an alkyl group or H, R₂ is H, CN, SO₃ ⁻, sulfamoyl, alkylsubstituted sulfamoyls, COO⁻, carbamoyl, alkyl substituted carbamoyls,R₃ is an alkyl group, and R₄ is an alkyl group.

In another embodiment, a nitric oxide probe of the present disclosurecomprises a compound that is represented by the following Formula II:

wherein R₁ is an alkyl group or H, R₂ is H, CN, SO₃ ⁻, sulfamoyl, alkylsubstituted sulfamoyls, COO⁻, carbamoyl, alkyl substituted carbamoyls,R₃ is an alkyl group, and R₄ is an alkyl group. In one particularembodiment, wherein R₁ is H, R₂ is CN, R₃ is an methyl group, and R₄ isan methyl group, the compound is referred to as NO₅₅₀.

In yet another embodiment, a nitric oxide probe of the presentdisclosure comprises a compound that is represented by the followingFormula III:

wherein R₁ is an alkyl group or H and R₂ is H, CN, SO₃ ⁻, sulfamoyl,alkyl substituted sulfamoyls, COO⁻, carbamoyl, alkyl substitutedcarbamoyls,.

In yet another embodiment, a nitric oxide probe of the presentdisclosure comprises a compound that is represented by the followingFormula IV:

wherein R₁ is an alkyl group or H and R₂ is H, CN, SO₃ ⁻, sulfamoyl,alkyl substituted sulfamoyls, COO⁻, carbamoyl, alkyl substitutedcarbamoyls.

In yet another embodiment, a nitric oxide probe of the presentdisclosure comprises a compound that is represented by the followingFormula V:

wherein R₁ is an alkyl group or H, R₂ is H, CN, SO₃ ⁻, sulfamoyl, alkylsubstituted sulfamoyls, COO⁻, carbamoyl, alkyl substituted carbamoylsand R₃ is an alkyl group or H.

In yet another embodiment, a nitric oxide probe of the presentdisclosure comprises a compound that is represented by the followingFormula VI:

wherein R₁ is an alkyl group or H, R₂ is H, CN, SO₃ ⁻, sulfamoyl, alkylsubstituted sulfamoyls, COO⁻, carbamoyl, alkyl substituted carbamoylsand R₃ is an alkyl group or H.

The features and advantages of the present invention will be readilyapparent to those skilled in the art upon a reading of the descriptionof the embodiments that follows.

DRAWINGS

Some specific example embodiments of the disclosure may be understood byreferring, in part, to the following description and the accompanyingdrawings.

FIG. 1 is an ortep drawing of AZO₅₅₀.

FIG. 2 is a graph depicting the kinetic profiles of the reactionsbetween NO₅₅₀ (10 μM) and NO (0.6 eq) at pH 7.4 (PBS buffer with 20%DMSO 50 mM) and pH 10 (carbonate buffer with 20% DMSO, 50 mM).

FIG. 3 is a graph depicting the absorption spectra of NO₅₅₀ and AZO₅₅₀.

FIGS. 4A is an overlay of the excitation spectra (λ_(em),=550 nm) andemission spectra (with λ_(ex)=470 nm) when different aliquots of NO areadded to a NO₅₅₀ solution (50 μM) in PBS buffer containing 20% DMSO.Spectra were collected 2 minutes after NO addition.

FIG. 4B is a graph depicting the enhancement of the fluorescenceintensity (λ_(ex)=470 nm and λ_(em)=550 nm) upon NO addition over aperiod of 2 minutes.

FIG. 4C is a graph depicting the kinetics profile of the reactionbetween NO₅₅₀ (λ_(em)=470 nm and λ_(em)=550 nm).

FIG. 4D is a chart depicting the fluorescence intensity of solutioncontaining NO₅₅₀ (50 μM) after addition of following substances: NO, andvarious equivalent of H₂O₂, NO₃ ⁻, NO₂ ⁻, ascorbic acid (AA),dehydroascorbic acid (DHA), ClO⁻, O₂ ⁻, OH⁻, O₃, ¹O₂, and ONOO⁻.

FIG. 5A is a graph depicting the emission spectra of the 20% DMSO in PBSbuffer (50 mM at pH 7.4 with 150 mM NaCl) and emission spectra of NO₅₅₀at 5 μM, 2.5 μM, and 1.25 μM respective. The excitation wavelength is470 nm.

FIG. 5B is a graph depicting the emission spectra of NO₅₅₀ at variousconcentrations corrected with the solvent background.

FIG. 5C is a graph depicting the averaged fluorescence intensity ofNO₅₅₀ in the range of 447 nm to 553 nm with excitation wavelength at 470nm.

FIG. 5D is a graph depicting the emission spectra of the 20% DMSO in PBSbuffer (50 mM at pH 7.4 with 150 mM NaCl) and emission spectra of AZO₅₅₀at 5 μM, 2.5 μM, and 1.25 μM respective. The excitation wavelength is470 nm.

FIG. 5E is a graph depicting the emission spectra of AZO₅₅₀ at variousconcentrations corrected with the solvent background.

FIG. 5F is a graph depicting the fluorescence intensity of AZO₅₅₀ at 550nm with excitation wavelength at 470 nm.

FIG. 5G is a graph depicting the ratio between the emission intensity ofAZO₅₅₀ and NO₅₅₀ at 550 nm with excitation wavelength at 470 nm.

FIG. 6 is a graph depicting the pH dependence of the fluorescenceintensity of AZO₅₅₀.

FIG. 7A is a phase contrast image (left) and corresponding wide-fieldfluorescence image (right) of neonatal spinal astrocytes stimulated withIFN-γ and IL-1β and incubated with 10 μM NO₅₅₀. Scale bar=150 μm. Linearlevels were adjusted using Adobe Photoshop to aide in visualization.

FIG. 7B is a phase contrast image (left) and corresponding wide-fieldfluorescence image (right) of astrocytes treated identically to those inFIG. 7A, except with the addition of 1 mM SNP. Scale bar=150 μm. Linearlevels were adjusted using Adobe Photoshop to aide in visualization.

FIG. 7C is a phase contrast image (left) and corresponding wide-fieldfluorescence image (right) of NGF-stimulated PC-12 cells incubated with10 μM NO₅₅₀. Scale bar=150 μm. Linear levels were adjusted using AdobePhotoshop to aide in visualization.

FIG. 7D is a phase contrast image (left) and corresponding wide-fieldfluorescence image (right) of NGF-stimulated PC-12 cells and imaged 10μM DAF-2 DA. Scale bar=150 μm. Linear levels were adjusted using AdobePhotoshop to aide in visualization.

FIG. 7E is a plot of fluorescence signal from raw images of astrocytesin FIGS. 7A and 7B. The plot represents a linescan constructed throughthe center (x-axis) of the images.

FIG. 7F is a plot of fluorescence signal from raw images of PC-12 cellsin FIGS. 7C and 7D. The plot represents a linescan constructed throughthe center (x-axis) of the images.

FIG. 8A-D depicts the imaging of NO using NO₅₅₀ at variousconcentrations in PC12 cells.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

While the present disclosure is susceptible to various modifications andalternative forms, specific example embodiments have been shown in thefigures and are described in more detail below. It should be understood,however, that the description of specific example embodiments is notintended to limit the invention to the particular forms disclosed, buton the contrary, this disclosure is to cover all modifications andequivalents as illustrated, in part, by the appended claims.

DESCRIPTION

The present disclosure generally relates to nitric oxide probes. Moreparticularly, the present disclosure relates to fluorescent nitric oxideprobes and associated methods.

The present disclosure provides a nitric oxide probe that may be used todetect and/or image NO. In one embodiment, a nitric oxide probe of thepresent disclosure comprises a compound that is represented by thefollowing Formula I:

wherein R₁ is an alkyl group or H, R₂ is H, CN, SO₃ ⁻, sulfamoyl, alkylsubstituted sulfamoyls, COO⁻, carbamoyl, alkyl substituted carbamoyls R₃is an alkyl group, and R₄ is an alkyl group.

In another embodiment, a nitric oxide probe of the present disclosurecomprises a compound that is represented by the following Formula II:

wherein R₁ is an alkyl group or H, R₂ is H, CN, SO₃ ⁻, sulfamoyl, alkylsubstituted sulfamoyls COO⁻, carbamoyl, alkyl substituted carbamoyls, R₃is an alkyl group, and R₄ is an alkyl group. In one particularembodiment, wherein R₁ is H, R₂ is CN, R₃ is a methyl group, and R₄ is amethyl group, the compound is referred to as NO₅₅₀.

In another embodiment, a nitric oxide probe of the present disclosurecomprises a compound that is represented by the following Formula III:

wherein R₁ is an alkyl group or H and R₂ is H, CN, SO₃ ⁻, sulfamoyl,alkyl substituted sulfamoyls, COO⁻, carbamoyl, alkyl substitutedcarbamoyls.

In another embodiment, a nitric oxide probe of the present disclosurecomprises a compound that is represented by the following Formula IV:

wherein R₁ is an alkyl group or H and R₂ is H, CN, SO₃ ⁻, sulfamoyl,alkyl substituted sulfamoyls, COO⁻, carbamoyl, alkyl substitutedcarbamoyls,.

In another embodiment, a nitric oxide probe of the present disclosurecomprises a compound that is represented by the following Formula V:

wherein R₁ is an alkyl group or H and R₂ is H, CN, SO₃ ⁻, sulfamoyl,alkyl substituted sulfamoyls, COO⁻, carbamoyl, alkyl substitutedcarbamoyls and R₃ is an alkyl group or H.

In another embodiment, a nitric oxide probe of the present disclosurecomprises a compound that is represented by the following Formula VI:

wherein R₁ is an alkyl group or H and R₂ is H, CN, SO₃ ⁻, sulfamoyl,alkyl substituted sulfamoyls, COO⁻, carbamoyl, alkyl substitutedcarbamoyls and R₃ is an alkyl group or H.

While not wishing to be bound to any particular theory, it is believedthat upon introduction of nitric oxide under aerobic conditions to anitric oxide probe of the present disclosure, a nitrosation reactionoccurs to yield a nitrosamine, which is subsequently scavenged via anelectronic aromatic substitution reaction. The product of this reactionhas an extended conjugation system in comparison to the initiallysynthesized compound and displays red shifted spectral properties, whichare easily detected through fluorescence.

One of the many advantages of the present disclosure, many of which arenot discussed herein, is that the nitric oxide probes of the presentdisclosure are highly selective and have not been found to interferewith reactive oxygenated species, reactive nitrogen species, ascorbicacid (AA), and dehydroascorbic acid (DHA). The probes of the presentdisclosure have also been shown to successfully respond to nitric oxidewithin cellular media. Due to the various roles of nitric oxide in thebody, a sensing method utilizing a nitric oxide probe of the presentdisclosure can be applied to study any of the biological pathways wherenitric oxide may be involved. In addition to the advantage of highspecificity, a nitric oxide probe of the present disclosure is alsoadvantageous due to its facile synthesis, low pH dependence, and fastreaction kinetics.

In one embodiment, a method of the present disclose comprises contactinga sample with a nitric oxide probe comprising a compound represented byFormula I-VI, or a combination thereof, and detecting emittedfluorescence from the nitric oxide probe. In some embodiments, thedetection of emitted fluorescence involves the detection of a turn onfluorescence signal from a dark background at the longer wavelength uponnitric oxide addition, rather than a fluorescent signal fluctuation froma non-zero background seen by most nitric oxide detecting systems.Highly electron rich ortho-diamino aromatics were avoided so as toimpede general oxidation by other reactive oxygen/nitrogen species andcondensation with ascorbic acid (AA) analogs. In some embodiments, aspectrofluorometer may be used to detect emitted fluorescence from anitric oxide probe.

Therefore, the present invention is well adapted to attain the ends andadvantages mentioned as well as those that are inherent therein. Whilenumerous changes may be made by those skilled in the art, such changesare encompassed within the spirit of this invention as illustrated, inpart, by the appended claims.

EXAMPLES

NO gas was bubbled into a solution of NO₅₅₀ in CH₂Cl₂. A color changefrom light yellow to deep red was observed spontaneously. Thisphenomenon qualitatively demonstrated that NO₅₅₀ is reactive towardNO/N₂O₃, and the structure of the product (AZO₅₅₀) was unambiguouslyestablished via NMR, MS, and X-ray crystallography (FIG. 1). The ringclosure occurs para to the dimethylamino group, not ortho, presumablydue to steric hindrance. The schematic illustration below depicts NO₅₅₀and the stepwise route to AZO₅₅₀.

The reaction between NO₅₅₀ and NO requires an NO⁺ equivalent, such asN₂O₃. The reaction resembles the synthesis of azo compounds via adiazotization/coupling sequence. However, a profound difference exists.Diazotization occurs in acidic media to form diazonium salts fromnitrosamines However, the reaction between NO₅₅₀ and NO occurs rapidlyat neutral (pH=7.4, vide infra) or even basic (pH=10, FIG. 2) aqueousconditions, where conversion of the initially formed nitrosaminederivative to a corresponding diazonium salt via acid-catalyzeddehydration is unlikely. This implies that electrophilic aromaticsubstitution on the electron deficient nitrosamine occurs to yield ahydroxyhydrazine derivative. Elimination of H₂O would lead to theformation of AZO₅₅₀. The lower intensity with the experiment at pH 10compared to that at pH 7.4 may be attributed to a combination of thefollowing factors: 1) reaction is slower, and 2) hydrolysis of N₂O₃ athigher pH is accelerated.

Due to the limited aqueous solubility of NO₅₅₀, 20% DMSO in PBS buffer(50 mM at pH=7.4 with 150 mM NaCl) was used for spectroscopic studies.NO₅₅₀ displays an absorption band centered at 352 nm which tails to 450nm (FIG. 3), and shows no fluorescence in aqueous media. It is believedthat the lack of emission is likely due to a combination of PET from the3′-dimethylaminophenyl ring and rotational deactivation along thearyl-aryl single bond. As an aliquot of the NO stock solution (1.9 mM inde-ionized H₂O) was added, an absorption band centered at 450 nmappeared immediately while that at 352 nm diminished. Concomitantly, anemission band centered at 550 nm (with a broad maximum excitationranging from 440 nm to 470 nm) appeared from a dark background (FIG.4A), and was identical to that for purified AZO₅₅₀. A completely darkbackground from which a bright signal appears in response to NO isunique for this strategy. A fluorescence enhancement over 1500 fold(FIG. 5) was observed compared to a ca. 200 fold enhancement with DAF-2DA (calculated with published photophysical parameters). Excellentlinear correlation (R²=0.995) was found between the fluorescenceenhancement and the NO concentration (FIG. 4B). The kinetics of thereaction of NO₅₅₀ with NO was studied via time-based fluorescence(λ_(ex)=470 nm and λ_(em)=550 nm). It took approximately 20 seconds forvarious aliquots of NO to complete the fast phase of the reaction, whichaccounts for 80% of overall signal enhancement. (FIG. 4C). This fastkinetic profile allows for good temporal resolution in cellular imagingstudies. Unlike the existing fluorescein-based probes, the fluorescenceof AZO₅₅₀ was independent of pH from 4.5 to 9 (FIG. 6), which isadvantageous because different cells or even the different compartmentsin the same cell may have different pH values. The molar absorptivityand quantum yield of AZO₅₅₀ were 4000 M⁻¹cm⁻¹ at 470 nm and 0.11respectively in 20% DMSO. The Φ is likely higher in the lipophilicregions of cells where NO is released. The limit of detection (LOD, whenthe signal equals three times the noise) was estimated to be around 30nM in 20% DMSO (FIG. 4B). A wide array of possible competitive reactiveoxygen or nitrogen species, and others analytes at up to 1000 foldexcess: H₂O₂, NO₃ ⁻, NO₂ ⁻, ascorbic acid (AA), dehydroascorbic acid(DHA), ClO⁻, O₂ ⁻, OH., O₃, ¹O₂, and ONOO⁻ (FIG. 4D) were also screened.None of these species induced any interfering signals due to our uniquedetection mechanism.

NO₅₅₀ was used to image NO in NSA and PC12 cell lines

NO₅₅₀ was used to visualize both endogenously produced NO, andexogenously supplied NO by an donor (sodium nitroprosside, SNP) in theculture of two cells types: neonatal spinal astrocytes and thepheochromocytoma-derived, PC12 cell line, which is commonly used as amodel for neurons. Both cell types have been previously reported toproduce NO. The NO₅₅₀ appears to enter the cytoplasm in both cell types,but does not cross into the nuclei. The viability of PC12 cells andastrocytes when incubated with the NO dye was evaluated by measuringtheir metabolic activity after 30 minutes. No differences were observedbetween cells incubated with the dye and controls.

Several in vitro experiments were performed to confirm that NO₅₅₀responded to NO in a biological context. Astrocytes were stimulated withinterleukin-1β (IL-1β) and interferon-γ (IFN-γ) to induce NO productionand then incubated with NO₅₅₀ for 15 minutes. A stronger fluorescencesignal was observed in the astrocytes that displayed the “spindle”phenotype, induced by cytokine stimulation, when compared to thoseexhibiting a flattened spread phenotype (FIG. 7A). This agrees withprevious reports that “spindle” phenotypes produce higher levels of NOwhen induced by cytokines. Longer incubation times (>15 minutes) withNO₅₅₀ did not lead to enhanced fluorescence, indicating that the probehad reached a diffusive equilibrium in the cells. Upon addition of an NOdonor (sodium nitroprusside, SNP) to cell cultures, a notable increasein fluorescence of both astrocytic phenotypes occurred (FIG. 7B, datawith PC12 cells not shown). A quantitative comparison of data from FIG.7A and 7B showed a measurably larger fuorescence increase when SNP isadded to the cell culture. (FIG. 7E)

NO₅₅₀ was also compared to a commercially available NO probe, DAF-2 DA.NO₅₅₀ yielded far less background fluorescence than DAF-2 DA whenincubated with PC12 cells and subsequently imaged (FIG. 7C,D,F). Thisresult is not unexpected, because DAF-2 itself hasbackground-fluorescence, and it reacts with dehydroascorbic acid, whichsignificantly contributes to the background. An even higher backgroundwas observed when DAR-4M AM was used (data not shown), which agrees withits elevated reactivity toward DHA compared to DAF-2 DA. In addition, ahigher concentration of NO₅₅₀ (up to 50 μM) could be used to furtherincrease the signal-to-background ratio in the images (FIG. 8). NO₅₅₀was not observed to permeate the nuclear membrane, while fluorescencewas observed in the cell nuclei in cultures incubated with DAF-2 DA. Thefluorescence signal from NO₅₅₀ was concentrated around the nuclei, whenincubated with both cell lines. This further confirms the utility ofNO₅₅₀ because NO synthase associates with the Golgi bodies near thenucleus in both cytokine-stimulated astrocytes and nerve growth factor(NGF) stimulated PC12 cells. Thus it is reasonable to expect that moreNO is present in these high density areas of NO synthase.

Synthetic scheme of NO₅₅₀ and AZO₅₅₀

Compound 1 was synthesized following literature procedures.Regioselective bromination of 1 (0.82 g) using NBS (0.91 g, 1.0 eq)yielded compound 2 (1.1 g, 91%) by stirring with a catalytic amount ofnormal phase silica gel (30 mg) in CH₃CN (100 mL) for 16 hrs at roomtemperature. The reaction was not protected from light or air. The crudeproduct after filtration and evaporation was purified by flash columnusing CH₂Cl₂ as eluent. ¹HNMR (300 MHz, DMSO-d₆) δ8.59 (d, 1H, J=8.7Hz), 8.10 (d, 1H, J=7.2 Hz), 7.72 (d, 1H, J=8.7 Hz), 7.22 (d, 1H, J=9.0Hz), 7.57 (dd, 1H, J=8.4, 7.5 Hz). ¹³CNMR (300 MHz, DMSO-d₆) δ142.7,133.3, 133.1, 131.9, 128.5, 124.3, 122.3, 117.8, 112.4, 108.8, 102.5.HRMS m/z=246.9871 (M+H⁺), calculated 246.9871.

The mixer containing 2 (95 mg), 3-dimethylaminophenylboronic acid (62mg, 1.0 eq), Pd(PPh₃)₄ (10 mg, 0.02 eq) and Na₂CO₃ (320 mg, 8.0 eq) in 8mL of H₂O:EtOH:Benzene (v/v 3:3:10) was refluxed for 24 hrs under argonatmosphere. The reaction mixture was poured into H₂O and extracted withCH₂Cl₂. The crude product after evaporation was purified by flash columnusing CH₂Cl₂ to yield NO₅₅₀ (106 mg, 99%). ¹HNMR (400 MHz, CDCl₃) δ8.13(d, 1H, J=8.8 Hz), 7.89 (dd, 1H, J=7.2, 1.2 Hz), 7.73 (dd, 1H, J=8.4,1.2 Hz), 7.54 (d, 1H, J=8.4 Hz), 7.50 (dd, 1H, J=8.8, 7.2 Hz), 7.38 (dd,1H, J=7.6, 7.6 Hz), 7.26 (s, 1H), 6.84-6.77 (m, 3H), 4.48 (s, 2H), 3.008(s, 6H). ¹³CNMR (400 MHz, CDCl₃) δ151.7, 140.5, 140.2, 133.4, 133.0,132.1, 130.5, 127.3, 125.4, 124.5, 123.9. 118.9, 118.0, 115.8, 113.9,112.4, 111.1, 41.2. HRMS m/z=288.1500 (M+H⁺), calculated 288.1495.

Bubbling NO gas into a CH₂Cl₂ solution containing NO₅₅₀ (13.5 mg) atroom temperature for 5 minutes yielded AZO₅₅₀ (14.0 mg, 100%) afterevaporation and flash column by CH₂Cl₂ with 1% ammonia saturated MeOH.¹HNMR (600 MHz, DMSO-d₆) δ9.86 (d, 1H, J=7.8 Hz), 8.95 (d, 1H, J=9.6Hz), 8.42 (d, 1H, J=9.6 Hz), 8.37 (dd, 1H, J=7.2, 1.2 Hz), 8.28 (dd, 1H,J=9.0, 1.0 Hz), 8.00 (dd, 1H, J=8.4, 7.2 Hz), 7.60 (dd, 1H, J=9.3, 2.4Hz), 7.56 (d, 1H, J=2.4 Hz), 3.25 (s, 6H). ¹³CNMR (600 MHz, DMSO-d₆)δ151.9, 142.0, 140.0, 134.3, 131.7, 131.6, 130.3, 129.1, 127.7, 125.8,123.7, 123.0, 118.7, 118.6, 117.5, 109.2, 97.6, 40.1. HRMS m/z=344.1147(M+H⁺), calculated 344.1148.

Materials and Methods

The ¹HNMR and ¹³CNMR spectra were recorded on Varian Unity Plus 300,Varian MERCURY 400 and Varian NOVA 600 spectrometers. High-resolution MS(HRMS) spectra were obtained a Micromass Autospec Ultima massspectrometer with a double focusing magnetic sector. UV-Vis spectra wererecorded on a Beckman Coulter DU 800 UV-Vis spectrophotometer.Fluorescence spectra were recorded on a PTI QuantaMaster™ intensitybased spectrofluorometer equipped with 814 photomultiplier detectionsystem (V=1000 volts). The excitation and emission slits were set at 1.0mm. All titrations were performed at aerobic conditions.

Purified neonatal astrocytes were obtained from PO rat spinal cords(Sprague Dawley, Charles Rivers, Wilmington, Mass.). All animal work wasperformed in accordance with the Institutional Animal Care and UseCommittee at the University of Texas at Austin. Astrocytes were culturedin DMEM (Sigma-Aldrich, St. Louis, Mo.) with 10% fetal bovine serum(Invitrogen, Carlsbad, Calif.) and 1% penicillin/streptomycin/amphotericin B (Sigma-Aldrich). Astrocyte cultures wereimmunostained against glial fibrillary acidic protein (GFAP, Abcam,Cambridge, Mass.) using standard procedures to confirm phenotype andused in experiments from passages 2-5. To induce NO production, cellswere stimulated with 200 U mL⁻¹ interferon-γ (IFN-γ) and 10 ng mL⁻¹interleukin-1β (IL-1β) for 24-48 h. PC12 cells were cultured inF12K/DMEM (Sigma-Aldrich) with 7.5% heat-inactivated house serum, 2.5%fetal bovine serum, and 1% penicillin/ streptomycin/amphotericin B. Toinduce NO production, PC12 cells were stimulated with 50 ng mL⁻¹ nervegrowth factor (NGF) for 8-10 days. Viability of cells incubated in NO₅₅₀was evaluated using a CellTiter Glo luminescence assay according to themanufacturer's instructions (Promega, Madison, Wis.).

Immediately prior to NO imaging, cells were rinsed in Dulbecco'sphosphate-buffered saline, pH 7.2 (PBS; Sigma-Aldrich) and kept in PBSduring the imaging procedure. The 10 μM of NO₅₅₀ or 10 μM ofdiaminofluorescein-2 (DAF-2 DA; Sigma-Aldrich); both diluted from stockin DMSO) was incubated with cells for approximately 15 min. at 37° C.before imaging. In some cases cells were incubated with 1 mM sodiumnitroprusside (Sigma-Aldrich) for 15 minutes before an additional rinsein PBS and incubation with NO₅₅₀. Phase contrasts and fluorescenceimages were acquired using a wide-field fluorescence microscope (IX70;Olympus; Center Valley, Pa.) equipped with a standard fluorescein filtercube set (Olympus) and a 20x (0.5 numerical aperture) objective(UPLFLN-PH, Olympus). Line plots of fluorescence signal were constructedusing ImageJ software (NIH, Bethesda, Md.).

The nitric oxide (NO) stock solution in de-ionized water was prepared bybubbling NO into deoxygenated de-ionized water for 15 minutes. (Ouyang,J. et al. A novel fluorescent probe for the detection of nitric oxide invitro and in vivo. Free Radic. Biol. Med. 45, 1426-1436 (2008)).Superoxide radical anion (O₂ ⁻) was from KO₂. Hydroxyl radical (HO.) wasgenerated from H₂O₂ and FeSO₄. Singlet oxygen (¹O₂) was generated fromClO⁻ and H₂O₂. Peroxynitrite was generated from amyl nitrite and H₂O₂following literature procedures. (Uppu, R. M. & Pryor, W. A. Synthesisof peroxynitrite in a two-phase system using isoamyl nitrite andhydrogen peroxide. Anal. Biochem. 236, 242-249, (1996)).

Therefore, the present invention is well adapted to attain the ends andadvantages mentioned as well as those that are inherent therein. Whilenumerous changes may be made by those skilled in the art, such changesare encompassed within the spirit of this invention as illustrated, inpart, by the appended claims.

References

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What is claimed is:
 1. A nitric oxide probe comprising a compoundrepresented by the following Formula I:

wherein R₁ is an alkyl group or H, R₂ is H, CN, SO₃ ⁻, sulfamoyl, alkylsubstituted sulfamoyls, COO⁻, carbamoyl, alkyl substituted carbamoyls,R₃ is an alkyl group, and R₄ is an alkyl group.
 2. A nitric oxide probecomprising a compound represented by the following Formula III:

wherein R₁ is an alkyl group or H, and R₂ is H, CN, SO₃ ⁻, sulfamoyl,alkyl substituted sulfamoyls, COO⁻, carbamoyl, alkyl substitutedcarbamoyls .
 3. A nitric oxide probe comprising a compound representedby the following Formula IV:

wherein R₁ is an alkyl group or H, and R₂ is H, CN, SO₃ ⁻, sulfamoyl,alkyl substituted sulfamoyls, COO⁻, carbamoyl, alkyl substitutedcarbamoyls.
 4. A nitric oxide probe comprising a compound represented bythe following Formula V:

wherein R₁ is an alkyl group or H, R₂ is H, CN, SO₃ ⁻, sulfamoyl, alkylsubstituted sulfamoyls, COO⁻, carbamoyl, alkyl substituted carbamoyls,and R₃ is an alkyl group or H.
 5. A nitric oxide probe comprising acompound represented by the following Formula VI:

wherein R₁ is an alkyl group or H, R₂ is H, CN, SO₃ ⁻, sulfamoyl, alkylsubstituted sulfamoyls, COO⁻, carbamoyl, alkyl substituted carbamoyls,and R₃ is an alkyl group or H.
 6. A method of detecting the presence ofnitric oxide in a sample comprising: contacting a sample with any of thenitric oxide probes of claims 1-5; and detecting emitted fluorescencefrom the nitric oxide probe, wherein the emitted fluorescence indicatesthe presence of nitric oxide in the sample.